The demands of an increasingly environmentally conscientious society and industries need to maximize its bottom line have driven a rapid increase in the use of enzymes and microorganisms for mediating industrially important chemical transformations (Schmid et al., Nature 409: 258-68, 2001; Ritter, Chem. Engin. News. 82:25-30, 2004). Generally, enzymes possess exceptional catalytic efficiencies, operate under very mild conditions (room temperature, neutral pH, ambient pressure), exhibit high selectivity and specificity, and generate minimal waste (Wong and Whitesides (1994). Enzymes in Synthetic Organic Chemistry. Oxford, Pergamon). As such, enzymes have the potential to favorably impact industrial chemical processes and provide an attractive alternative to traditional chemical synthesis.
Several nitrile metabolizing enzymes have been identified and used in biocatalysis (FIG. 1). For example, the use of nitrile hydratase (NHase), which converts nitrites to amides, in the industrial synthesis of acrylamide represented the first application of biocatalysis to commodity chemical synthesis (Kobayashi et al., Trends Biotech. 10:402-8, 1992), and the first use of biocatalysis in the petroleum industry. Other commercial applications of NHase include the production of nicotinamide from 3-cyanopyridine (Mathew et al., Appl. Environ. Microbiol. 54:1030-2, 1988), the production of benzamide and thiophenamide from the corresponding nitrites, and the conversion of the nitrile groups in acrylic fibers to the corresponding amides (Tauber et al., Appl. Environ. Microbiol. 66:1634-8, 2000).
The enzyme nitrilase, which hydrolyzes nitrites to carboxylic acids, has also been incorporated into many commercial processes. For example, the vitamins nicotinic acid and p-aminobenzoic acid have been prepared from the nitrilase-catalyzed hydrolysis of 3-cyanopyridine and p-aminobenzonitrile, respectively. Nitrilase and NHase are also used as agents in the bioremediation of nitrile containing waste streams and in herbicide degradation (Banerjee et al., Appl. Environ. Micro. 60:33-44, 2002).
One area where biocatalysis has yet to impact the commercial reactions of nitrites is in their reduction to primary amines. This is due to the lack of any known enzyme capable of carrying out the reduction of nitrites to amines (FIG. 1). The reduction of nitrites to amines has traditionally been carried out by hydrogenation over various transition metal catalysts or by metal hydride reductions (March, J. (1992). Advanced Organic Chemistry: Reactions, Mechanisms, and Structure. New York, John Wiley & Sons). These reactions are typically non-selective, requiring the use of protecting groups when other reducible functional groups are present, and can result in the formation of unwanted byproducts.
Thus, identification of an enzyme that is capable of reducing nitrites to amines would permit biocatalysis of this reaction, and provide an alternative to the synthetic conversion of nitrites to amines.